Protein synthesis is a fundamental biological process, which underlies the development of polypeptide therapeutics, diagnostics, and catalysts. With the advent of recombinant DNA (rDNA) technology, it has become possible to harness the catalytic machinery of the cell to produce a desired protein. This can be achieved within the cellular environment or in vitro using extracts derived from cells.
Over the past decade, the productivity of cell-free systems has improved two orders of magnitude, from about 5 μg/ml-hr to about 500 μg/ml-hr. This accomplishment has made in vitro protein synthesis a practical technique for laboratory-scale research and provides a platform technology for high-throughput protein expression. It also begins to suggest the feasibility of using cell-free technologies as an alternative means to the in vivo large-scale production of protein pharmaceuticals.
Cell-free protein synthesis offers several advantages over conventional, in vivo, protein expression methods. Cell-free systems can direct most, if not all, of the metabolic resources of the cell towards the exclusive production of one protein. Moreover, the lack of a cell wall in vitro is advantageous since it allows for better control of the synthesis environment. For example, tRNA levels can be changed to reflect the codon usage of genes being expressed. Also, the redox potential, pH, or ionic strength can be altered with greater flexibility than in vivo since cell growth or viability is not a concern. Furthermore, direct recovery of purified, properly folded protein products can be easily achieved.
In vitro translation is also recognized for its ability to incorporate unnatural and isotope-labeled amino acids as well as its capability to produce proteins that are unstable, insoluble, or cytotoxic in vivo. In addition, cell-free protein synthesis may play a role in revolutionizing protein engineering and proteomic screening technologies. The cell-free method bypasses the laborious processes required for cloning and transforming cells for the expression of new gene products in vivo, and is becoming a platform technology for this field.
Despite all of the promising features of cell-free protein synthesis, its practical use and large-scale implementation has been limited by several obstacles. Paramount among these are short reaction times and low protein production rates, which lead to poor yields of protein synthesis and excessive reagent cost. The pioneering work of Spirin et al. (1988) Science 242:1162-1164 initially circumvented the short reaction times problem with the development of a continuous flow system. Many laboratories have duplicated and improved upon this work, but they have all primarily used methods that constantly supply substrates to the reaction chamber. This approach increases the duration of the translation reaction and protein yield as compared to the batch system. However, it is inefficient in its use of expensive reagents, generally produces a dilute product, and has not provided significant improvements in production rates.
The conventional batch system offers several advantages over these continuous and semi-continuous schemes, which include ease of scale-up, reproducibility, increased protein production rates, convenience, applicability for multi-plexed formats for high throughput expression, and more efficient substrate use. These advantages make improving the batch system productivity crucial for the industrial utilization of cell-free protein synthesis. However, using current methodology, when reactions are scaled up there is a loss of efficiency. The decrease in specific protein product yield is especially severe in the systems that require oxygen for oxidative phosphorylation. Increasing the product yield in larger reactions is an essential component of filling this need.
Relevant Literature
U.S. Pat. No. 6,337,191 B1, Swartz et al. Kim and Swartz (2000) Biotechnol Prog. 16:385-390; Kim and Swartz (2000) Biotechnol Lett. 22:1537-1542; Kim and Choi (2000) J Biotechnol. 84:27-32; Kim et al. (1996) Eur J Biochem. 239: 881-886; Tao and Levy (1993) Nature 362:755-758; Hakim et al. (1996) J Immun. 157:5503-5511; Pratt (1984) Coupled transcription-translation in prokaryotic cell-free systems. In: Hames B D, Higgins S J. Ed. In transcription and translation: a practical approach. New York: IRL press: 179-209.; Davanloo et al. (1984) PNAS 81:2035-2039; Cock et al. (1999) Biochemistry 259: 96-103; Gill and Hippel (1989) Anal. Biochem. 182:319-326; Kim et al. (1999) Europ. J. Biochem. 239: 881-886; Davanloo et al. (1984) PNAS 81:2035-2039